DNA ligases are critical enzymes for virtually all DNA transactions, including DNA replication, repair and recombination. Vertebrates have three DNA ligase genes: Lig1, Lig3 and Lig4; none can be directly deleted from the mouse germline. The Lig3 gene encodes two isoforms: mitochondrial Lig3 (which is cell essential) and the more abundant nuclear Lig3. Recently, we established a mutant mouse strain harboring a knock-in mutation that specifically ablates nuclear Lig3 using a one-step CRISPR/Cas9- mediated genome editing strategy in mouse embryos. Nuclear Lig3 is widely considered to be the primary ligase for DNA single strand break (SSB) repair due to its strong interaction with an essential SSBR factor called X-ray cross complementation factor 1 (XRCC1). Our unique mouse model will allow for the first time, a thorough dissection of the function of Lig3 in nuclear DNA repair in vivo. Experiments proposed in this application will provide the initial characterization of these mice. A comprehensive pathological phenotype analysis will be performed. Because DNA repair defects are often associated with genomic instability and neurodegenerative diseases, additional experiments will focus on the impact of Lig3 deficiency on spontaneous tumor development and neuropathology. Successful completion of the proposed studies will provide unprecedented insight into Lig3's normal physiologic role. Conversely, Lig3 overexpression is found in a number of tumor cell lines and tumor samples from human patients; this impacts DNA double strand break (DSB) repair such that the Lig4-dependent canonical non-homologous end-joining (NHEJ) is, to varying extents, replaced by the Lig3-dependent alternative end-joining (A-EJ), which has been implicated in chromosomal translocations. We will address the impact of nuclear Lig3 deficiency on IgH/c-myc translocation that occurs during immunoglobulin (Ig) heavy (H) chain class switch recombination (CSR). These particular translocations are critical to the development of many human and mouse B cell tumors. Furthermore, characterizing the dependence of these translocations on Lig3 may be relevant to many other tumor types. Successful completion of these studies may elucidate a pathological role of Lig3 in tumorigenesis.